The invention relates to drug-screening assays for evaluating and identifying compounds capable of affecting pathogenicity and virulence of a pathogen.
Pathogens employ a number of genetic strategies to cause infection and, occasionally, disease in their hosts. The expression of microbial pathogenicity is dependent upon complex genetic regulatory circuits. Knowledge of the themes in microbial pathogenicity is necessary for understanding pathogen virulence mechanisms and for the development of new “anti-virulence” or “anti-pathogenic” agents, which are needed to combat infection and disease.
The mechanism of pathogenesis and the host defense is a field of intense investigation. Antibiotics have been an effective tool to treat unwanted bacterial infections. However, due to the increasing incidence of resistance to current antibiotics, new antibiotics are needed. Antibiotics that target non-essential genes are desirable because there is limited, if any, selection pressure on these genes since they are not required for the survival of the bacteria. Thus, bacteria are less likely to develop resistance to antibiotics that target these genes.
In nature, most bacteria live not as individual cells but as pseudo-multicellular organisms that coordinate their population behavior via small extracellular signal molecules. Under appropriate conditions, these molecules are released into the environment, taken up, and responded to by surrounding cells (Fuqua et al., Annu. Rev. Genet. 35, 439-68, 2001; Miller et al., Annu. Rev. Microbiol. 55, 165-199, 2001; Withers et al., Curr. Opin. Microbiol. 4, 186-193, 2001). ‘Quorum sensing’ (QS), is the archetypal intercellular communication system used by many bacterial species to regulate their gene expression in response to cell density. Using this regulatory system, all the individual bacterial cells behave coordinately and synergistically as a community, for example, in growth dynamics and resource utilization (Fuqua et al., J. Bacteriol. 176, 269-275, 1994). A common feature of all QS systems is the transcriptional activation and repression of a large regulon of QS-controlled genes when a minimal threshold concentration of a specific autoinducer is reached.
The QS system used by Gram-negative bacteria is mediated by the extracellular signaling molecules, N-acyl-L-homoserine lactones (AHLs) (Withers et al., Curr Opin Microbiol 4, 186-193, 2001; Fuqua et al., Annu Rev Genet 35: 439-68, 2001). The versatile and ubiquitous opportunistic pathogen Pseudomonas aeruginosa is one of the best-studied models of AHL-mediated QS. In this species, two separate autoinducer synthase/transcriptional regulator pairs, LasRI and RhlRI, modulate the expression of several genes, including many virulence factors, in response to increasing concentrations of the specific signaling molecules oxo-C12—HSL and C4—HSL (Pesci et al., in Cell-cell signaling in bacteria, eds., 1999; Van Delden et al., Emerg. Infect. Dis. 4, 551-560, 1998).
P. aeruginosa also produces a cell-to-cell signal distinct from AHLs: 3,4-dihydroxy-2-heptylquinoline, called the PQS signal (Pesci et al., Proc. Natl. Acad. Sci. USA 96, 11229-11234, 1999). PQS serves as a signaling molecule regulating the expression of a subset of genes belonging to the QS regulon, including the phz and hcn operons. PQS functions in the QS hierarchy by linking a regulatory cascade between the las and the rhl systems (McKnight et al., J. Bacteriol. 182, 2702-2708, 2000). That maximal PQS production occurs at the end of the exponential growth phase (Lépine et al., Biochim. Biophys. Acta 1622, 36-40, 2003) supports the hypothesis that PQS acts as a secondary regulatory signal for a subset of QS-controlled genes. Although PQS has no antibiotic activity (Pesci et al., Proc. Natl. Acad. Sci. USA 96, 11229-11234, 1999), it belongs to a family of poorly characterized antimicrobial P. aeruginosa products, the ‘pyo’ compounds, originally described in 1945, which are derivatives of 4-hydroxy-2-alkylquinolines (HAQs) (Hays et al., J. Biol. Chem. 159, 725-750, 1945; Wells, J. Biol. Chem. 196, 331-340, 1952). The QS-associated P. aeruginosa transcriptional regulator, MvfR, is also required for the production of several secreted compounds, including virulence factors and PQS (Cao et al., Proc. Natl. Acad. Sci. USA 98, 14613-8, 2001; Rahme et al., Proc. Natl. Acad. Sci. USA 94, 13245-13250, 1997). MvfR controls the synthesis of anthranilic acid (AA), a PQS precursor (Calfee et al., Proc. Natl. Acad. Sci. USA 98, 11633-11637, 2001), by positively regulating the transcription of phnAB, which encodes an anthranilate synthase (Cao et al., Proc. Natl. Acad. Sci. USA 98, 14613-8, 2001).
This pathway represents a candidate target for the pharmacological intervention of P. aeruginosa mediated infections.